The global biologics market has steadily growth in recent years. Among the biologics, the top product has been monoclonal antibodies (mAb), achieving sales of $16.9 billion in the US in 2009 (Aggarwal S 2010. Nature Biotechnology 28(11):1165-1171). There are currently over 20 mAbs approved for sale and almost a hundred more going through various stages of clinical testing (Nelson A L, Dhimolea E, Reichert J M. 2010. Nat Rev Drug Discov 9(10):767-74). Mammalian cells, particularly Chinese hamster ovary (CHO) cells are the dominant host for industrial mAb production because of their capacity for proper protein folding, assembly and appropriate post-translational modifications (Wurm F M 2004. Nat Biotechnol 22(11):1393-8).
To obtain high-level expression, the most commonly used expression system is dihydrofolate reductase deficient (DHFR−) CHO cells in conjunction with two vectors, one expressing a DHFR selection marker linked to one mAb gene, light chain (LC) or heavy chain (HC), the other vector expressing an alternative selection marker linked to the second mAb gene (Kim N S, Kim S J, Lee G M. 1998a. Biotechnology And Bioengineering 60(6):679-688; Kim S J, Kim N S, Ryu C J, Hong H J, Lee G M. 1998b. Biotechnol Bioeng 58(1):73-84.). Each gene is under the control of its own promoter and expressed as separate transcript. DHFR catalyzes the reduction of dihydrofolate into tetrahydrofolate, an essential co-factor in the synthesis of purines and amino acids.
To enrich the proportion of high producers after drug selection thus reduce efforts of clone screening, the stringency of clone selection can be improved by weakening the selection marker. The principle is that only those clones with greater transcription activity or more copies of the integrated vector, which often means higher productivity, survives the selection process (Fussenegger, M et al., 1999. Trends In Biotechnology 17(1): 35-42; Ng, S K et al. 2007. Metabolic Engineering 9(3): 304-316).
In the context of DHFR based selection systems, the expression is performed in media lacking glycine, hypoxanthine, and thymidine and enhanced expression level can be achieved by exposure to methotrexate (MTX), a DHFR inhibitor, leading to the amplification the gene copy numbers. Theoretically, after a few rounds of selection in medium containing stepwise increments of methotrexate (MTX), the copies of the gene of interest should be increased up to hundred folds leading to clones with high productivity.
Other reported strategies of weakening selection marker are based on either reducing expression level through application of weak regulatory elements like promoters on the selection marker (Barnett, R S et al. 1995. Antibody Expression And Engineering. 604: 27-40.) or use of codon deoptimized selection marker (Westwood, A D et al. 2010. Biotechnology Progress 26(6): 1558-1566), or impairment of the selection marker function, such as mutation of critical amino acids in neomycin phosphorase (NPT) selection marker (Yenofsky, R L et al. 1990. PNAS 87(9): 3435-3439; Niwa, H et al. 1991. Gene 108(2): 193-199; Sautter, K and Enenkel, B 2005. Biotechnol Bioeng 89(5): 530-538).
However, the above technologies are limited by the fact that, e.g., due to vector fragmentation with concomitant deletion of the mAb gene expression cassettes effective simultaneous amplification of both antibody chains is rare. In many cases, only the mAb chain linked to the marker is amplified or even none of both mAb genes is amplified. As a result, a large number of clones have to be screened to obtain high producing clones, making the cell line generation process labor intensive and extremely time consuming.
Internal ribosome entry site (IRES) and 2A peptide provides alternative approaches for co-expression of multiple genes. Internal ribosome entry site (IRES) elements allow expression of multiple genes in one transcript (Mountford and Smith 1995). IRES-based bicistronic or tricistronic vectors, which express the product and selection marker genes in one transcript, can minimize the escape of non-expressing clones from selection, as none of the two genes will be expressed should vector fragmentation happen. In these vectors, the product and selection marker genes are under the control of the same promoter. However, one concern for the use of IRES in expressing mAbs is that the gene driven by IRES has lower translation efficiency than the gene under the 5′-cap dependent translation.
In contrast, the 2A peptide allows translation of multiple proteins in a single open reading frame into a polyprotein that is subsequently cleaved into individual proteins through a ribosome-skipping mechanism (Funston, Kallioinen et al. 2008). As compared to IRES, 2A peptide may provide more balanced expression of LC and HC. However, the large size of the polyprotein, e.g., the sum of LC and HC, is believed to result in lower translation efficiency and product instability.
So far, there are very few studies on the use of IRES and 2A for the generation of stable recombinant expressing cell lines.
There is still need in the art for nucleic acid molecules encoding for recombinant peptides or proteins and comprising 2A and/or IRES sequences that allow for an improved production of highly productive and/or stable cell lines.